LOCUS       sequence-331760-        7830 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  use as an auxiliary temperature-sensitive plasmid to successfully 
            transform pdCas9_CL plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7830)
  AUTHORS   Huang HH, Bellato M, Qian Y, Cardenas P, Pasotti L, Magni P, Del 
            Vecchio D
  TITLE     dCas9 regulator to neutralize competition in CRISPRi circuits.
  JOURNAL   Nat Commun. 2021 Mar 16;12(1):1692. doi: 10.1038/s41467-021-21772-6.
  PUBMED    33727557
REFERENCE   2  (bases 1 to 7830)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-03-16"; volume: "12(1):1692. doi"; pages: " 
            10.1038/s41467-021-21772-6"
COMMENT     Sequence Label: pAUX_OL
FEATURES             Location/Qualifiers
     source          1..7830
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        81..156
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    211..232
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        233..267
                     /label=BBa_J23116
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.16"
     RBS             276..287
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(294..320,321..323,324..2810,2811..2813,2814..4400)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     terminator      4424..4495
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4511..4538
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      4556..4800
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             complement(4923..5873)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      5921..6143
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(6765..7556,7557..7625))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7388..7407
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7626..7730)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(7817..7830,1..5)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gaggcagggc gcgccaaaaa gagtattgac ttcgcatctt tttgtaccta taatagattc
       61 agtattgact attaatcatc gttttagagc tagaaatagc aagttaaaat aaggctagtc
      121 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttccaatta ttgaacaccc
      181 taacgggtgt tttttttttt ttggtctccc gaattcgcgg ccgcttctag agttgacagc
      241 tagctcagtc ctagggacta tgctagctac tagagaaaga ggagaaagga tctatggata
      301 agaaatactc aataggctta gctatcggca caaatagcgt cggatgggcg gtgatcactg
      361 atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac cgccacagta
      421 tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg gaagcgactc
      481 gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt tgttatctac
      541 aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat cgacttgaag
      601 agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt ggaaatatag
      661 tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga aaaaaattgg
      721 tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg catatgatta
      781 agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt gatgtggaca
      841 aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac cctattaacg
      901 caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca agacgattag
      961 aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg aatctcattg
     1021 ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca gaagatgcta
     1081 aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg gcgcaaattg
     1141 gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct attttacttt
     1201 cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct tcaatgatta
     1261 aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt cgacaacaac
     1321 ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat gcaggttata
     1381 ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt ttagaaaaaa
     1441 tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg cgcaagcaac
     1501 ggacctttga caacggctct attccccatc aaattcactt gggtgagctg catgctattt
     1561 tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag attgaaaaaa
     1621 tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat agtcgttttg
     1681 catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa gaagttgtcg
     1741 ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat aaaaatcttc
     1801 caaatgaaaa agtactacca aaacatagtt tgctttatga gtattttacg gtttataacg
     1861 aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa accagcattt ctttcaggtg
     1921 aacagaagaa agccattgtt gatttactct tcaaaacaaa tcgaaaagta accgttaagc
     1981 aattaaaaga agattatttc aaaaaaatag aatgttttga tagtgttgaa atttcaggag
     2041 ttgaagatag atttaatgct tcattaggta cctaccatga tttgctaaaa attattaaag
     2101 ataaagattt tttggataat gaagaaaatg aagatatctt agaggatatt gttttaacat
     2161 tgaccttatt tgaagatagg gagatgattg aggaaagact taaaacatat gctcacctct
     2221 ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg ggacgtttgt
     2281 ctcgaaaatt gattaatggt attagggata agcaatctgg caaaacaata ttagattttt
     2341 tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct gatccatgat gatagtttga
     2401 catttaaaga agacattcaa aaagcacaag tgtctggaca aggcgatagt ttacatgaac
     2461 atattgcaaa tttagctggt agccctgcta ttaaaaaagg tattttacag actgtaaaag
     2521 ttgttgatga attggtcaaa gtaatggggc ggcataagcc agaaaatatc gttattgaaa
     2581 tggcacgtga aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag cgtatgaaac
     2641 gaatcgaaga aggtatcaaa gaattaggaa gtcagattct taaagagcat cctgttgaaa
     2701 atactcaatt gcaaaatgaa aagctctatc tctattatct ccaaaatgga agagacatgt
     2761 atgtggacca agaattagat attaatcgtt taagtgatta tgatgtcgat gccattgttc
     2821 cacaaagttt ccttaaagac gattcaatag acaataaggt cttaacgcgt tctgataaaa
     2881 atcgtggtaa atcggataac gttccaagtg aagaagtagt caaaaagatg aaaaactatt
     2941 ggagacaact tctaaacgcc aagttaatca ctcaacgtaa gtttgataat ttaacgaaag
     3001 ctgaacgtgg aggtttgagt gaacttgata aagctggttt tatcaaacgc caattggttg
     3061 aaactcgcca aatcactaag catgtggcac aaattttgga tagtcgcatg aatactaaat
     3121 acgatgaaaa tgataaactt attcgagagg ttaaagtgat taccttaaaa tctaaattag
     3181 tttctgactt ccgaaaagat ttccaattct ataaagtacg tgagattaac aattaccatc
     3241 atgcccatga tgcgtatcta aatgccgtcg ttggaactgc tttgattaag aaatatccaa
     3301 aacttgaatc ggagtttgtc tatggtgatt ataaagttta tgatgttcgt aaaatgattg
     3361 ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac tctaatatca
     3421 tgaacttctt caaaacagaa attacacttg caaatggaga gattcgcaaa cgccctctaa
     3481 tcgaaactaa tggggaaact ggagaaattg tctgggataa agggcgagat tttgccacag
     3541 tgcgcaaagt attgtccatg ccccaagtca atattgtcaa gaaaacagaa gtacagacag
     3601 gcggattctc caaggagtca attttaccaa aaagaaattc ggacaagctt attgctcgta
     3661 aaaaagactg ggatccaaaa aaatatggtg gttttgatag tccaacggta gcttattcag
     3721 tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc gttaaagagt
     3781 tactagggat cacaattatg gaaagaagtt cctttgaaaa aaatccgatt gactttttag
     3841 aagctaaagg atataaggaa gttaaaaaag acttaatcat taaactacct aaatatagtc
     3901 tttttgagtt agaaaacggt cgtaaacgga tgctggctag tgccggagaa ttacaaaaag
     3961 gaaatgagct ggctctgcca agcaaatatg tgaatttttt atatttagct agtcattatg
     4021 aaaagttgaa gggtagtcca gaagataacg aacaaaaaca attgtttgtg gagcagcata
     4081 agcattattt agatgagatt attgagcaaa tcagtgaatt ttctaagcgt gttattttag
     4141 cagatgccaa tttagataaa gttcttagtg catataacaa acatagagac aaaccaatac
     4201 gtgaacaagc agaaaatatt attcatttat ttacgttgac gaatcttgga gctcccgctg
     4261 cttttaaata ttttgataca acaattgatc gtaaacgata tacgtctaca aaagaagttt
     4321 tagatgccac tcttatccat caatccatca ctggtcttta tgaaacacgc attgatttga
     4381 gtcagctagg aggtgactaa ctcgagtaag gatctccagg catcaaataa aacgaaaggc
     4441 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta
     4501 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tataaatggc gatgacgcat
     4561 cctcacgata atatccgggt aggcgcaatc actttcgtct actccgttac aaagcgaggc
     4621 tgggtatttc ccggcctttc tgttatccga aatccactga aagcacagcg gctggctgag
     4681 gagataaata ataaacgagg ggctgtatgc acaaagcatc ttctgttgag ttaagaacga
     4741 gtatcgagat ggcacatagc cttgctcaaa ttggaatcag gtttgtgcca ataccagtag
     4801 aaacagacga agaatccatg ggtatggaca gttttccctt tgatatgtaa cggtgaacag
     4861 ttgttctact tttgtttgtt agtcttgatg cttcactgat agatacaaga gccataagaa
     4921 cctcagatcc ttccgtattt agccagtatg ttctctagtg tggttcgttg tttttgcgtg
     4981 agccatgaga acgaaccatt gagatcatac ttactttgca tgtcactcaa aaattttgcc
     5041 tcaaaactgg tgagctgaat ttttgcagtt aaagcatcgt gtagtgtttt tcttagtccg
     5101 ttatgtaggt aggaatctga tgtaatggtt gttggtattt tgtcaccatt catttttatc
     5161 tggttgttct caagttcggt tacgagatcc atttgtctat ctagttcaac ttggaaaatc
     5221 aacgtatcag tcgggcggcc tcgcttatca accaccaatt tcatattgct gtaagtgttt
     5281 aaatctttac ttattggttt caaaacccat tggttaagcc ttttaaactc atggtagtta
     5341 ttttcaagca ttaacatgaa cttaaattca tcaaggctaa tctctatatt tgccttgtga
     5401 gttttctttt gtgttagttc ttttaataac cactcataaa tcctcataga gtatttgttt
     5461 tcaaaagact taacatgttc cagattatat tttatgaatt tttttaactg gaaaagataa
     5521 ggcaatatct cttcactaaa aactaattct aatttttcgc ttgagaactt ggcatagttt
     5581 gtccactgga aaatctcaaa gcctttaacc aaaggattcc tgatttccac agttctcgtc
     5641 atcagctctc tggttgcttt agctaataca ccataagcat tttccctact gatgttcatc
     5701 atctgaacgt attggttata agtgaacgat accgtccgtt ctttccttgt agggttttca
     5761 atcgtggggt tgagtagtgc cacacagcat aaaattagct tggtttcatg ctccgttaag
     5821 tcatagcgac taatcgctag ttcatttgct ttgaaaacaa ctaattcaga catacatctc
     5881 aattggtcta ggtgatttta atcactatac caattgagat gggctagtca atgataatta
     5941 ctagtccttt tcctttgagt tgtgggtatc tgtaaattct gctagacctt tgctggaaaa
     6001 cttgtaaatt ctgctagacc ctctgtaaat tccgctagac ctttgtgtgt tttttttgtt
     6061 tatattcaag tggttataat ttatagaata aagaaagaat aaaaaaagat aaaaagaata
     6121 gatcccagcc ctgtgtataa ctcactactt tagtcagttc cgcagtatta caaaaggatg
     6181 tcgcaaacgc tgtttgctcc tctacaaaac agaccttaaa accctaaagg cttaagtagc
     6241 accctcgcaa gctcggttgc ggccgcaatc gggcaaatcg ctgaatattc cttttgtctc
     6301 cgaccatcag gcacctgagt cgctgtcttt ttcgtgacat tcagttcgct gcgctcacgg
     6361 ctctggcagt gaatgggggt aaatggcact acaggcgcct tttatggatt catgcaagga
     6421 aactacccat aatacaagaa aagcccgtca cgggcttctc agggcgtttt atggcgggtc
     6481 tgctatgtgg tgctatctga ctttttgctg ttcagcagtt cctgccctct gattttccag
     6541 tctgaccact tcggattatc ccgtgacagg tcattcagac tggctaatgc acccagtaag
     6601 gcagcggtat catcaacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6661 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6721 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6781 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6841 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6901 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6961 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     7021 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     7081 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     7141 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7201 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7261 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7321 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7381 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7441 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7501 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7561 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7621 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7681 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7741 cgaaaagtgc cacctgcatc gatttatgcc acctgacgtc taagaaacca ttattatcat
     7801 gacattaacc tataaaaata ggcgtatcac
//
